Haemophilus somnus is a Gram negative bacterium which causes a number of disease syndromes in animals. The bacterium is commonly associated with thromboembolic meningoencephalitis (ITEME), septicemia, arthritis, and pneumonia (Corbeil, L. B., Can. J. Vet. Res. (1990) 54:S57-S62; Harris, F. W., and Janzen, E. D., Can. Vet. J. (1990) 30:816-822; Humphrey, J. D., and Stephens, L. R., Vet. Bull. (1983) 53:987-1004). These diseases can cause significant economic losses to the farm industry.
Currently available vaccines are either based on killed whole cells or on a protein fraction enriched in outer membrane proteins (OMPs). However, whole cell bacterins and surface protein extracts often contain immunosuppressive components which can render animals more susceptible to infection. Furthermore, the OMP enriched vaccine has only been shown to offer significant protection against H. somnus induced disease in an experimental challenge model (Harland, R. J., et al., Res. Work. Anim. Dis. 71st (1990) 29:6).
The outer membrane of H. somnus includes a 40 kDa protein (as determined by SDS-PAGE) which reacts with convalescent serum (Corbeil, L. B., et al., infect. Immun. (1987) 55:1381-1386; Goglolewski, R. P., et al., Infect. Immun. (1988) 56:2307-2316). Additionally, antibodies directed against a 40 kDa OMP have been shown to prevent infection in vitro in a neutralization experiment (Gogolewski et al., supra) and a seroreactive protein of 40 kDa is present in all H. somnus isolates that have been tested (Corbeil et al., 1987).
A 39 kDa OMP, antigenically distinct from the 40 kDa OMP described above, has also been identified. This protein reacts with convalescent-phase serum and is conserved among all H. somnus isolates tested.
An increasing number of bacterial antigens have now been identified as lipoproteins (Anderson, B. E., et al., J. Bacteriol. (1988) 170:4493-4500; Bricker, T. M., et al., Infect. Immun. (1988) 56:295-301; Hanson, M. S., and Hansen, E. J., Mol. Microbiol. (1991) 5:267-278; Hubbard, C. L., et al., Infect. Immun. (1991) 59:1521-1528; Nelson, M. B., et al., Infect. Immun. (1988) 56:128-134; Thirkell, D., et al., Infect. Immun. (1991) 59:781-784). These lipoproteins are generally localized in the envelope of the cell and are therefore exposed to the host's immune system. It has been shown that the murein lipoprotein from the outer membrane of Escherichia coli acts as a potent activator of murine lymphocytes, inducing both proliferation and immunoglobulin secretion (Bessler, W., et al. Z. Immun. (1977) 153:11-22; Melchers, F., et al. J. Exp. Med. (1975 142:473-482). The active lipoprotein portion of the protein has been shown to reside in the N-terminal fatty acid containing region of the protein. Recent studies using synthetic lipopeptides based on this protein show that even short peptides, containing two to five amino acids covalently linked to palmitate, are able to activate murine lymphocytes (Bessler, W. G., et al. J. Immunol. (1985) 135:1900-1905).
To date, only one such lipoprotein from H. somnus has been positively identified. This protein, termed "LppA", is an OMP with an apparent molecular mass of 40 kDa, as determined by gel electrophoresis. The nucleotide sequence for LppA has been determined (Theisen, M., et al., Infect. Immun. (1992) 60:826-831). However, the protective capability of this protein has not previously been studied.